Keel bone fractures affect welfare, health, and production performance in laying hens. A total of one hundred and twenty 35-wk-old Hy-line Brown laying hens with normal keel (NK) bone were housed in furnished cages and studied for ten weeks to investigate the underlying mechanism of keel bone fractures. At 45 wk of age, the keel bone state of birds was assessed by palpation and X-ray, and laying hens were recognized as NK and fractured keel (FK) birds according to the presence or absence of fractures in keel bone. The serum samples of 10 NK and 10 FK birds were collected to determine bone metabolism-related indexes and slaughtered to collect keel bones for RNA-sequencing (RNA-seq), Micro-CT, and histopathological staining analyses. The results showed that the concentrations of Ca, phosphorus, calcitonin, 25-hydroxyvitamin D3, and osteocalcin and activities of alkaline phosphatase and tartrate-resistant acid phosphatase (TRAP) in serum samples of FK birds were lower than those of NK birds (P 1, P < 0.05), among which 88 were upregulated and 126 downregulated. Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) analysis indicated that 14 DEGs related to skeletal muscle movement and bone Ca transport (COL6A1, COL6A2, COL6A3, PDGFA, MYLK2, EGF, CAV3, ADRA1D, BDKRB1, CACNA1S, TNN, TNNC1, TNNC2, and RYR3) were enriched in focal adhesion and Ca signaling pathway, regulating bone quality. This study suggests that abnormal bone metabolism related to keel bone fractures is possibly responded to fracture healing in laying hens.